Nutrient Remineralization by Ramicrusta

Background

Brief Introduction:

The rapid proliferation of the red encrusting macroalgal Ramicrusta in the Caribbean is raising concerns about its ecological impacts, particularly its ability to outcompete foundational benthic species, inhibit invertebrate recruitment, and overgrow living coral colonies, often leading to coral mortality (Eckrich & Engel, 2013; Edmunds et al., 2019). Coral genera exhibit varying levels of susceptibility to Ramicrusta colonization, with Orbicella sp. among the most vulnerable and experiencing high alga overgrowth rates (Hollister et al., 2021; Fig. 1).

There’s been considerable evidence illustrating Ramicrusta’s tendency to interact with and overgrow live corals (Eckrich & Engel, 2013; Ballantine et al., 2016; Edmunds et al., 2019; Hollister et al., 2021), yet there remains a disconnect when it comes to understanding the factors contributing to this relationship. As macroalgal growth is generally restricted by nutrient availability, particulary in these oligotrophic water, I hypothesize that Ramicrusta may be deriving nutrients from the coral itself, fueling these interactions and promoting its own growth.

 

Fig. 1: R. textilis overgrowing an O. annularis colony (photo: Abigail Gretta).

 

Application of Stable Isotope Analysis:

Based on predictable isotope fractionation patterns (Fry, 1988), the ability of the alga to remineralize coral nutrients can be evaluated through bulk stable isotope analyses. Fractionation refers to the preferential use of the lighter isotope (¹²C, ¹⁴N) during metabolic processes, concentrating the heavier form in the organism’s tissues (¹³C, ¹⁵N) (Rounick & Winterbourn, 1986; Fry, 1988; Cohen & Fong, 2004). In heterotrophic consumers, the ratio of heavy to light isotopes for carbon (C) and nitrogen (N) generally enrich by 1‰ and 3‰ per trophic level, respectively (Fry, 1988). These ratios, or isotope signatures, are represented by the delta notation in parts per thousand (\(\delta^{13}\)C, \(\delta^{15}\)N). This unique aspect of isotope fractionation provides insight into a consumer’s food source (\(\delta^{13}\)C) and trophic position (\(\delta^{15}\)N) (Rounick & Winterbourn, 1986; Post et al., 2002). However, primary producers typically exhibit a lack of fractionation (Gartner et al., 2002; Cohen & Fong, 2004; Strait et al., 2021). Thus, both the C and N signatures generally reflect their source. As a result, macroalgal bioassays serve as powerful proxies for nutrient studies in oligotrophic environments because tissue analyses can trace nutrient sourcing (\(\delta^{13}\)C, \(\delta^{15}\)N), availability (C%, N%), and limitations (C:N) in the surrounding water (Amato et al., 2016; Amato et al., 2018; Strait et al., 2021).

In theory, if Ramicrusta species are remineralizing coral tissues, the C and N isotope signatures of the algae should mirror those of the coral (Cohen & Fong, 2004). Additionally, the C and N percentages may be enhanced in the alga as a result of additional coral nutrients, which would, in turn, reduce nutrient limitations and lower the C:N ratio. (Amato et al., 2016). However, baseline tissue nutrient analyses on the genus Ramicrusta are lacking; therefore, we will need to include two upright geniculate calcifying macroalgae species into our sampling to offer context into Ramicrusta’s nutrient parameters. The rhizophytic green alga Halimeda opuntia and epilithic red alga Jania adhereans will be prioritized because they are prominent calcifiers within the region and their nutrient contents have been established for oligotrophic reef systems (Fong et al., 2003; Koch et al., 2023).

The following are the preliminary methods, results, and conclusions from a pilot study conducted on September 3, 2024.

Methods

Site Description:

Sample collection occurred on the leeward side of Flat Cay, an offshore 9 m depth reef along the southwest side of St. Thomas, U.S. Virgin Islands (Fig. 2). This site was selected because of its high abundance of R. textilis (46.0% ± 5.9; Hollister et al., 2021) and high frequency of interactions between the alga and Orbicella annularis (personal observation). To characterize the abiotic conditions during algal and coral sampling, a temperature logger (Hobo Pendant) and PAR meter were deployed a month prior to the collection time and retrieved on the final day of sampling.

Fig. 2: Algal collection occurred on September 2, 2024 on the leeward side of Flat Cay, St. Thomas, a 9 m depth aggregated reef. Effects of remineralization may have been masked by the high nitrogen content found in all alga species, thus Fortuna Bay and Flat Cay windward were marked as future resampling sites because of the considerably low growth rates of R. textilis on bare substrate when compared to O. annularis at these locations (Hollister et al., 2021).

Alga Collection:

Open-circuit SCUBA was used for all algal collections. Replicates of each algal sample was randomly collected by hand, with a minimum of five meters between samples. The apical regions of H. opuntia and J. adhaerens (~ 5 cm) were cleanly removed from its thallus (n = 10 samples per species). R. textilis was collected from ten rocks (control; n = 10) and ten O. annualaris colonies that appeared visually healthy. For each substrate, R. textilis was brushed of epiphytes and chiseled at two locations from the same contiguous alga thallus (n = 2 R. textilis samples per substrate). One sample was collected from the margin of algal growth and the other was at a linear distance away from the margin, with distances between samples measured (~ 2 to 3 cm) and photographed (Fig. 3). This sampling strategy will distinguish the alga’s nutrient parameters during direct interaction with living coral to those in non-interacting regions, with the algal samples from bare substrate serving as controls for both conditions. Algal replicates will be placed in individual plastic bags at depth with ambient seawater. Samples will be transported to the laboratory in a dark container to minimize physiological stress and processed immediately.

Fig. 3: R. textilis were collected from 10 O. annularis colonies (left) and 10 rocks (control), with marginal and non-marginal replicates sampled from each substrate type (n = 2 algal samples per substrate).

Isotope Preparation:

Macroalgae were rinsed with deionized water (DI) to remove any external contaminants (i.e., invertebrates and epiphytes) and thoroughly dried with paper towels (Strait et al., 2021). Samples were loosely parceled in combusted aluminum foil with each sample code and dried at 60℃ to constant weight in a drying oven (24-36 hours). With a clean mortar and pestle (ethanol and DI rinse between samples), algae were ground to a fine powder and carefully packaged in 1.5 mL Eppendorf tubes (Strait et al., 2021). Stable isotope analyses were processed by an Elemental Analyzer Delta V at the University of Hawai’i at Mānoa. Dual stable isotope analyses were performed because acidification has the potential to degrade \(\delta^{15}\)N and unaltering calcified tissue can skew \(\delta^{13}\)C (Strait & Spalding, 2021).

Data Analysis:

All data were tested for homogeneity and normality. Log transformations were used when data violated ANOVA assumptions. Tissue nutrients (\(\delta^{15}\)N, % N, and C:N) were assessed using two-factor ANOVA (factors: Substrate, Growth Region, and the interaction of the two). Tukey’s honestly significant difference post hoc test was applied to show pairwise comparisons, with distinct letters on graphs denoting significant differences.

Results

Effects of Remineralization:

A two-factor ANOVA with a log transformation revealed significant differences in Ramicrusta’s \(\delta^{15}\)N for substrate type (p-value = 0.04978) and growth region (p-value = 0.01112), with no significant interaction effect between the two factors. Marginal (mean \(\delta^{15}\)N ± SEM = 1.66 ‰ ± 0.10) and non-marginal (mean \(\delta^{15}\)N ± SEM = 1.81 ‰ ± 0.06) samples of R. textilis growing on O. annularis had higher N signatures compared to their corresponding controls (mean \(\delta^{15}\)N ± SEM = 1.36 ‰ ± 0.13 and 1.78 ‰ ± 0.13, respectively; Fig. 5). Nitrogen signatures were consistently lower in marginal samples than in non-marginal samples across both substrates. In contrast, the N percentages showed an opposite pattern for each substrate, with marginal samples from O. annularis colonies having significantly higher N percentages than non-marginal samples, while no significant difference was observed between the two regions on bare substrate (Fig. 6). The C:N ratio was significantly lower on O. annularis colonies (mean of C:N ± SEM = 15.21 ± 0.47) when compared to bare substrate (mean of C:N ± SEM = 17.72 ± 0.57), with no difference was detected for growth region or the interaction of the main effects (Fig. 7).

 

Calcifying Macroalgae Isotopes:

The N percentages across all calcifying macroalgae species exceeded 2% (Fig. 8), suggesting potential nutrient enrichment at the site (Amato et al., 2016). However, the N isotopic signatures, ranging from 0.7‰ to 2.4‰, indicate that the N inputs likely originated from non-anthropogenic sources (Strait et al., 2021).

 

Fig. 5: Boxplot of the N signatures from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between substrate (p-value = 0.04978) and growth region (p = 0.01112), but not the interaction effect of the two factors. Letters denote significant differences from a Tukey’s post hoc test.

 

Fig. 6: Boxplot of the % N from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between growth region (p-value = 0.0005287), but not between substrate types or the interaction effect of the two factors. Letters denote significant differences from a Tukey’s post hoc test.

 

Fig. 7: Boxplot of the C:N from R. textilis growing on O. annularis (n = 10 colonies) and bare substrate (i.e., control; n = 10 rocks), with a marginal and non-marginal samples collected from each substrate type. A two-factor ANOVA found significant differences between substrate (p-value = 0.00223), but not between the growth regions or the interaction effect of the two factors. Letters denote significant differences from a two-factor ANOVA.

 

Fig. 8: Boxplot of the % N across all calcifying macroalgal species. The high N content among all calcifers suggests nutrient enrichment may be a confounding factor at this location.

 

Brief Conclusion:

The significantly higher N signatures combined with the greater disparity between the marginal and non-marginal N percentages in the alga overgrowing O. annularis colonies suggests that Ramicrusta may possess the ability to remineralize coral-derived nutrients during overgrowth. This finding supports the hypothesis of a nutrient exchange dynamic between the alga and its coral host. However, the consistently high N percentages across all sampled calcifying macroalgae suggest that the study site may be experiencing nutrient enrichment, potentially obscuring the specific effects of this interaction. To address this, additional sampling will be conducted at two newly selected sites in early December to further investigate and clarify these nutrient dynamics.

References

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Misc. Analysis

\(\delta^{15}\)N

Analysis of Variance Model

	Df	Sum Sq	Mean Sq	F value	Pr(>F)
Substrate	1	0.1896	0.1896	4.158	0.04978
Growth_Region	1	0.3313	0.3313	7.264	0.01112
Substrate:Growth_Region	1	0.08185	0.08185	1.794	0.1898
Residuals	32	1.46	0.04562	NA	NA

% N

Analysis of Variance Model

	Df	Sum Sq	Mean Sq	F value	Pr(>F)
Substrate	1	0.49	0.49	0.8808	0.355
Growth_Region	1	8.258	8.258	14.84	0.0005287
Substrate:Growth_Region	1	1.491	1.491	2.681	0.1114
Residuals	32	17.8	0.5563	NA	NA

C:N

Analysis of Variance Model

	Df	Sum Sq	Mean Sq	F value	Pr(>F)
Substrate	1	56.95	56.95	11.05	0.00223
Growth_Region	1	0.6441	0.6441	0.125	0.726
Substrate:Growth_Region	1	0.03711	0.03711	0.007202	0.9329
Residuals	32	164.9	5.153	NA	NA